1. Area of the Art
The present invention relates to methods and apparatus for assaying biomolecular characteristics and structural configurations effective for use within the context of microfluidic loading and transfer methods. Particularly, the present invention relates to novel enhanced techniques for the facilitation of fabrication and improvement of microapparatus including the development of a microfabricated PCR reactor that is integrated on a CE chip, inter alia.
2. Description of the Prior Art
The "DNA Chip" has been heralded as the long awaited union between contemporary microelectronic technology and the genetic engineering arts. (Wade, N., "Meeting of Computers and Biology: The DNA Chip", Apr. 8, 1997, Science Times, THE NEW YORK TIMES, N.Y. Times New Service). The present invention constitutes another aspect of the innovative cycle which was both the genesis of contemporary `gene chip` technology and a prominent candidate for its most utile application to date.
The Polymerase Chain Reaction ("PCR") is a powerful procedure for amplifying and labeling long stretches of DNA using chromosomal or plasmid DNA as well as labeled nucleotides, those skilled in the art define the same as an in vitro technique for rapidly synthesizing large quantities of a given DNA segment that involves separating the DNA into its two complementary strands, binding a primer to each single strand at the end of the given DNA segment where synthesis will start, using DNA polymerase to synthesize two-stranded DNA from each single strand, and repeating the process.
Likewise, Capillary Electrophoresis ("CE") is a method of using silica capillaries to separate a wide variety of solutes, both charged and uncharged, and having particularly effective uses for the separation of small peptides, proteins and the like biomolecular moities, as in the case of the instant teachings.
By way of background, attention is called to co-pending U.S. Ser. No. 08/535,875; filed Sep. 28, 1995, which is assigned a common assignee, and was likewise invented by one of the present inventors. The '875 app. is incorporated in its entirety herein by reference, and provides valuable insight into the state of the art.
Generic schemata which likewise comprise basic reactor designs, including miniature temperature controlled reaction chambers for carrying out a variety of synthetic and diagnostic applications, for tasks from sizing of nucleic acids for hybridization, chemical labeling, thermal cycling, nucleic acid fragmentation, sizing and transcription all of the way to rudimentary sequencing were disclosed by the '875 app.
A large number of diagnostic and synthetic chemical reactions require repeated cycling through a number of specific temperatures to carry out the melting, annealing, and ligation or extension steps which are part of the respective processes. By reducing reaction volumes, the amount of time required for thermal cycling may also be reduced, thereby accelerating the amplification process. Further, this reduction in volume also results in a reduction of the amounts of reagents and sample used, thereby decreasing costs and facilitating analyses of increasingly smaller amounts of material.
Similarly, in hybridization applications, precise temperature controls likewise are used to obtain optimal hybridization conditions. Finally, a number of other pre- and post-hybridization treatments also require some degree of precise temperature control, such as fragmentation, transcription, chain extension for sequencing, labeling, ligation reactions, and the like.
A number of researchers have attempted to miniaturize and integrate reaction vessels for carrying out a variety of chemical reactions, including nucleic acid manipulation. For example, published PCT Application No. WO 94/05414, to Northrup and White reports an integrated micro-PCR apparatus fabricated from thin silicon wafers, for collection and amplification of nucleic acids from a specimen. Similarly, U.S. Pat. No. 5,304,487 to Wilding, et al., and U.S. Pat. No. 5,296,374 to Kricka, et al. discuss chambers and flow channels micromachined from silicon substrates for use in conjunction with the collection and analysis of cell samples. However, neither of these references address the added constraints of PCR, and how to protect adequately against the same.
The increased desire for automated chemical processes in both analytical and synthetic applications has led to a need for further miniaturization and integration of existing processes and equipment for carrying out such processes.
For miniaturized DNA analysis the successful and reliable coupling of PCR amplification and electrophoretic DNA separation constitutes a particularly noteworthy and certainly laudable aspiration. Integration of such techniques offers numerous potential advantages in terms of speed, cost and automation. Multiple reactors and separation channels have been envisaged by artisans as part of a high throughput genetic analysis system. The first effort which has become generally accepted among the technical community as a reasonable attempt at a substantially integrated PCR reactor on a CE chip was the hybrid device reported by Woolley and coworkers in 1996 (Woolley, A. T., Hadley, D., Landre, P., deMello, A. J., Mathies, R. A. and Northrup, M A., 1996, 68 Analytical Chemistry 4081-4086) as a part of a collaboration likewise initiated by a NIST ATP Project.
Woolley's team integrated a silicon sandwiched PCR type of reactor fabricated by Northrup and coworkers at LLNL with a glass CE chip fabricated at UC Berkeley. The Si reactor was mated to a side channel through a polypropylene sleeve. An HEC sieving matrix was used as an electrophoretic valve to separate the PCR solutions from the CE channels.
Much later, Ramsey and coworkers simply placed the PCR mix in a plastic reservoir on the chip and thermally cycled the entire chip on a conventional cycler. This was followed by injection as done earlier. This cycler was much slower than the hybrid device developed in the Berkeley-LLNL collaboration but did lead to ostensibly successful amplifications (Waters, L. C., Jacobson, S. C., Kroutchinina, N., Khandurina, J., Foote, R. S. and Ramsey, J. M., 1998, 70 (1) Anal. Chem. 158-162).
By way of further background, attention is likewise called to the following U.S. Pat. Nos: 20 4,821,997; 5,241,363; 5,554,276; 5, 585,069; 5,593,838; 5,603,351; 5,632,876; 5,643,738; and, 5,681,484.
It is respectfully submitted that each of the cited references merely defines the state of the art, or highlights aspects of the problems addressed and ameliorated according to the teachings of the present invention. The same are also fully referenced, and upon review each is clearly distinguished, as will be seen from the IDS filed concurrently herewith. Accordingly, further discussions of these references has been omitted at this time due to the fact that each of the same is readily distinguishable from the instant teachings to one having a modicum of skill in the art, as shall be denoued by the claims which are appended hereto.
To date the present inventors are not aware of any successful efforts to fabricate a fully integrated monolithic small volume PCR-CE device in glass using thin film metal heaters and thermocouples to thermally cycle sub-microlitre PCR volumes. Accordingly it is the longstanding need to address and ameliorate this concern which is a primary focus of the teachings of the present invention.